1. Field of the Invention
This invention relates to a process for producing a remodeled carbohydrate or glycoconjugate by utilizing the transglycosylation activity of a glycosidase.
2. Description of Related Art
Sugar chains of glycoproteins play important roles in various biological phenomenon, such as the immune second-messenger system. Therefore, modification or replacement of sugar chains in glycoproteins is useful for the pharmacological industry.
This technique can create new types of drugs that can be better delivered to the target cells or can have longer activity. Two approaches can be used for this purpose: chemical and enzymatic. Since chemical reactions usually require organic mediums or other harsh conditions, this approach may cause denaturation of glycoproteins. On the other hand, an enzymatic approach is usually carried out in aqueous solutions such as buffer solutions, etc., and thus are not damaging the proteins. Another advantage of an enzymatic method is that it will react more specifically.
The techniques for enzymatically remodeling sugar chains which are generally employed these days involve an exoglycosidase or a glycosyltransferase and sequential conversion starting from the nonreducing end of a sugar chain, as reported by D. H. Joziasse et al., European Journal of Biochemistry, 191, 75-83 (1990).
In regard to transglycosylation by an endoglycosidase, R. B. Trimble et al. Journal of Biological Chemistry, 261, 12000-12005 (1986)! have reported one relating to Endo-.beta.-N-acetylglucosaminidase F (Endo-F) from Flavobacterium meningosepticum and R. M. Bardales et al. Journal of Biological Chemistry, 264, 19893-19897 (1989)! have reported another one relating to endo-.alpha.-N-acetylgalactosaminidase from Diplococcus pneumoniae. Glycerol serves as an acceptor in the former report, while glycerol, tris, p-nitrophenol, serine and threonine serve as an acceptor in the latter report.
Reports on a remodeling mechanism, wherein a carbohydrate or a glycoconjugate serves directly as an acceptor, include one relating to Endo-.beta.-N-acetylglucosaminidase A (Endo-A) from Arthrobacter protophormiae disclosed by Japanese Patent Laid-Open No. 64594/1993 and one relating to Endo-.beta.-N-acetylglucosaminidase M (Endo-M) from Mucor hiemalis described in Seikagaku, 65, 1014 (1993).
When a glycoconjugate with a remodeled sugar chain is improved in the stability and biological activities compared with the corresponding natural glycoconjugate, it is highly applicable to drugs, as described above.
However, it is necessary in the conventional remodeling techniques in the presence of exoglycosidases or glycosyltransferases to perform an enzymatic reaction for each sugar residue, which requires a number of reaction steps and highly complicated procedures.
Also, neither a carbohydrate nor a glycoconjugate serves as an acceptor in the above-mentioned transglycosylation with the use of Endo-F or endo-.alpha.-N-acetylgalactosaminidase from Diplococcus pneumoniae.
On the other hand, the transglycosylation in the presence of Endo-A or Endo-M is a remodeling reaction wherein a carbohydrate or a glycoconjugate serves directly as an acceptor. Thus it is the most convenient and practically useful method for remodeling sugar chains known at the present time.